Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: Comparison of reconstituted vesicles with native membrane vesicles
Abstract
A Na+-specific and Na+-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na+ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K+, Li+, and choline+. The electrochemical effect of the lipophilic anion, SCN−, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl− or SO42−. The Na+-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent Km of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na+-specific and Na+-stimulated concentrative α-aminoisobutyric acid uptake.
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Cited by (2)
Effectors of amino acid transport processes in animal cell membranes
1985, Comparative Biochemistry and Physiology -- Part A: Physiology- 1.
1. Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions.
- 2.
2. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it.
- 3.
3. Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl− Both H+ and K+ have been found to be inhibitory in other systems.
- 4.
4. Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl− and H+ gradient or an outwardly directed K+ gradient can energize transport.
- 5.
5. Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient. Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport.
- 6.
6. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes.
- 7.
7. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis.
- 8.
8. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.
- 9.
9. Catecholamines directly stimulate amino acid transport through alpha-adrenergic mediation by a process which involves protein synthesis.
- 10.
10. Protein hormone secretagogues such as caerulein inhibit amino acid transport by a process that is mediated by Ca2+ .
- 11.
11. Cyclic nucleotides have been shown to have both stimulatory and inhibitory effects on amino acid transport, depending on the cell system. Some studies suggest that protein synthesis is involved in the stimulation by cyclic nucleotides, although in one cell system protein synthesis was ruled out as a cause of the stimulation. Stimulation of phosphorylation of membrane components by a cyclic nucleotide may be a mechanism to regulate amino acid transport.
- 12.
12. Glycoprotein glycosylation appears to be required for amino acid transport. Recent results suggest that a glycoprotein component of system A must be continually synthesized to sustain an increase in transport activity.
- 13.
13. Microfilament accumulation which occurs as a compact network beneath the cell membrane in some systems is responsible for inhibition of amino acid transport.
- 14.
14. Beta-adrenergic agonism effected by isoproterenol evokes a Ca2+-dependent stimulation of amino acid transport and an associated increase in polyamine levels (e.g. putrescine).
- 15.
15. Cell membrane thiol groups as well as cytosolic ones appear to be involved in amino acid transport.
- 16.
16. Gamma-glutamyl transpeptidase appears to be involved in the transport of a variety of amino acids in animal cell systems.
- 17.
17. The inhibitory effects on amino acid transport of trypsin, polyanions (e.g. dextran sulfate and heparin), phloretin, retinol, bile salts, pyridoxal phosphate, platinum co-ordination complexes, nicotine and ethanol and the stimulatory effects of polycations (e.g. neomycin), phorbol ester, serum albumin and indomethacin are described in this report.
- 1.
Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae
1982, BBA - General Subjectsα-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a Km for α-aminoisobutyric acid of 270 μM, a Vmax of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a Vmax of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH4+. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.